Simpson D A, Feeney S, Boyle C, Stitt A W
Department of Ophthalmology, The Queen's University of Belfast, The Royal Victoria Hospital, Belfast BT12 6BA, Northern Ireland, UK.
Mol Vis. 2000 Oct 5;6:178-83.
To determine whether continuous monitoring of SYBR Green I fluorescence provides a reliable and flexible method of quantitative RT-PCR. Our aims were (i) to test whether SYBR Green I analysis could quantify a wide range of known VEGF template concentrations, (ii) to apply this method in an experimental model, and (iii) to determine whether 20 existing primer pairs could be used to quantify their cognate mRNAs.
Real-time quantitative PCR was performed using a LightCycler rapid thermal cycler (Roche). Retinal VEGF mRNA levels were measured in a murine model of oxygen-induced retinopathy during vaso-obliterative and hypoxic phases.
This technique was able to detect as few as 10 control template copies, with quantitative data available routinely for 1000 or more copies. The levels of retinal VEGF mRNA expression followed the hypoxia-induced pattern determined previously by conventional methods. All gene-specific primer pairs which amplify a specific product by conventional PCR were successfully converted to SYBR Green analysis, including those for housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), cyclophilin, and acidic ribosomal phosphoprotein PO (ARP/36B4) and for 28S rRNA. In each case melting curve analysis and agarose gel electrophoresis confirmed the specificity of the amplification product.
The sequence-independent detection of DNA with SYBR Green I means that it can be used to quantify the amplification of any cDNA using gene-specific primers. This rapid and flexible method is ideally suited for researchers in vision science wishing to quantify mRNAs from many different genes because it does not require investment in gene-specific hybridization probes.
确定对SYBR Green I荧光进行连续监测是否能提供一种可靠且灵活的定量逆转录聚合酶链反应（RT-PCR）方法。我们的目标是：（i）测试SYBR Green I分析是否能对广泛的已知血管内皮生长因子（VEGF）模板浓度进行定量；（ii）在一个实验模型中应用该方法；（iii）确定20对现有的引物对是否可用于定量其同源信使核糖核酸（mRNA）。
使用罗氏LightCycler快速热循环仪进行实时定量PCR。在氧诱导性视网膜病变的小鼠模型中，于血管闭塞期和缺氧期测量视网膜VEGF mRNA水平。
该技术能够检测低至10个对照模板拷贝，常规可获得1000个或更多拷贝的定量数据。视网膜VEGF mRNA表达水平遵循先前通过传统方法确定的缺氧诱导模式。所有通过常规PCR扩增特定产物的基因特异性引物对都成功转化为SYBR Green分析，包括用于看家基因甘油醛-3-磷酸脱氢酶（GAPDH）、亲环蛋白和酸性核糖体磷蛋白PO（ARP/36B4）以及28S rRNA的引物对。在每种情况下，熔解曲线分析和琼脂糖凝胶电泳均证实了扩增产物的特异性。
SYBR Green I对DNA进行的序列非依赖性检测意味着它可用于使用基因特异性引物对任何互补DNA（cDNA）的扩增进行定量。这种快速且灵活的方法非常适合视觉科学领域希望对来自许多不同基因的mRNA进行定量的研究人员，因为它不需要投资购买基因特异性杂交探针。
